ly-341495 and Glioma

We have characterised, using both in vivo and in vitro methods, the effects of the metabotropic glutamate receptor subtype 3 (mGlu3) antagonist (LY341495) and agonist (LY379268) on the proliferation and differentiation of glioma stem cells (GSC). For in vitro studies, a CCK-8 assay was used to determine the cell proliferation, flow cytometry was performed to determine cell cycle phases, and immunohistochemistry and laser confocal microscopy were employed to detect CD133 expression. For in vivo studies, GSCs were injected into nude mice treated with either LY379268 or LY341495 and the growth of the tumours was measured after 3 weeks. When compared with controls, the proliferation rates and proportion of cells in S phase within the LY341495 treated group decreased in a time-dependent manner. In the presence of differentiation medium containing LY341495, GSC differentiation was diverted into an astrocyte rather than neuronal phenotype. The growth rate and volume of tumours injected into nude mice was reduced in LY341495 treated mice compared with controls. Thus pharmacological blockade of mGlu3 receptor signalling pathway significantly inhibits the growth and proliferation of GSCs both in vitro and in vivo while promoting differentiation to astrocytes. These results further implicate mGlu3 in the biology of glioma and as a target for continued research.

Topics: AC133 Antigen; Amino Acids; Animals; Antigens, CD; Astrocytes; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Glycoproteins; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Peptides; Phenotype; Receptors, Metabotropic Glutamate; Recombinant Proteins; Transplantation, Heterologous; Xanthenes

Targeted-therapies enhancing differentiation of glioma-initiating cells (GICs) are potential innovative approaches to the treatment of malignant gliomas. These cells support tumour growth and recurrence and are resistant to radiotherapy and chemotherapy. We have found that GICs express mGlu3 metabotropic glutamate receptors. Activation of these receptors sustained the undifferentiated state of GICs in culture by negatively modulating the action of bone morphogenetic proteins, which physiologically signal through the phosphorylation of the transcription factors, Smads. The cross-talk between mGlu3 receptors and BMP receptors was mediated by the activation of the mitogen-activated protein kinase pathway. Remarkably, pharmacological blockade of mGlu3 receptors stimulated the differentiation of cultured GICs into astrocytes, an effect that appeared to be long lasting, independent of the growth conditions, and irreversible. In in vivo experiments, a 3-month treatment with the brain-permeant mGlu receptor antagonist, LY341495 limited the growth of infiltrating brain tumours originating from GICs implanted into the brain parenchyma of nude mice. While clusters of tumour cells were consistently found in the brain of control mice, they were virtually absent in a large proportion of mice treated with LY341495. These findings pave the way to a new non-cytotoxic treatment of malignant gliomas based on the use of mGlu3 receptor antagonists.

Topics: Amino Acids; Animals; Bone Morphogenetic Protein Receptors; Brain Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Humans; Magnetic Resonance Imaging; Mice; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Phosphorylation; Receptors, Metabotropic Glutamate; Signal Transduction; Xanthenes

U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.

Topics: Amino Acids; Animals; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclins; Enzyme Activation; Excitatory Amino Acid Antagonists; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Magnetic Resonance Imaging; MAP Kinase Signaling System; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; Xanthenes

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.

Topics: Amino Acids; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cells, Cultured; Cyclin D1; Cyclin D2; Cyclins; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Receptors, Metabotropic Glutamate; Thymidine; Tritium; Xanthenes